CN110819635B - Application of HAN homologous gene of leguminous plant in regulating and controlling number of root nodules of leguminous plant - Google Patents
Application of HAN homologous gene of leguminous plant in regulating and controlling number of root nodules of leguminous plant Download PDFInfo
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- CN110819635B CN110819635B CN201911067613.6A CN201911067613A CN110819635B CN 110819635 B CN110819635 B CN 110819635B CN 201911067613 A CN201911067613 A CN 201911067613A CN 110819635 B CN110819635 B CN 110819635B
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The invention discloses an application of a legume HAN homologous gene in regulating the number of legume nodules, wherein the legume HAN homologous gene is selected from MtHAN1 gene and MtHAN2 gene, and the nucleotide sequence is shown as SEQ ID No.1 and 2. The invention separates out the knockout mutant strain of the MtHAN1 and MtHAN2 genes of the medicago truncatula for the first time, and finds that the root nodule number of the double mutant strain is obviously increased. This demonstrates that this gene and its homologues in other legumes are involved in regulating nodule production in legumes, affecting nodule numbers. The gene is knocked out, the transgenic leguminous plant with the root nodule number higher than that of a wild plant can be obtained, and the application of the gene is indicated to create a novel leguminous plant with less application of nitrogen fertilizer and higher utilization efficiency of the nitrogen fertilizer, so that the gene can be used for subsequent improvement of the quality of pasture and the yield of grain, and has great significance for the grass industry and the grain production in China.
Description
Technical Field
The invention relates to application of a HAN homologous gene of leguminous plants in regulation and control of the number of root nodules of the leguminous plants, belonging to the technical field of genetic engineering.
Background
Root nodules are the enlarged tissue that rhizobia interact with the roots of legumes to produce on the roots of plants. During the symbiosis process, the rhizobia fixes free nitrogen in the air into nitrogen-containing compounds for the plants to utilize by utilizing minerals and energy sources provided by the plants. The rhizobia and plants keep balance in the antagonistic relationship and mutually benefit and live in symbiosis.
Nitrogen is an essential nutrient for plants and is one of the most used fertilizers in crop fertilization. China is a big agricultural country and a big population country, and under the background of more population and less cultivated land, the crop yield must be improved to meet the grain demand, so the application amount of nitrogen fertilizer in China is increased year by year. According to statistics, the application amount of nitrogen fertilizer in China is less than 1000 ten thousand tons in the early 80 th of 19 th century, but the usage amount of nitrogen fertilizer in China breaks through 2000 ten thousand tons in the middle 90 th of 19 th century, and the nitrogen fertilizer tends to increase year by year. Although the application amount of the nitrogen fertilizer is rapidly increased, the utilization efficiency of the nitrogen fertilizer of the plants is not increased and is always stabilized to be about 35 percent, and the loss of a large amount of the nitrogen fertilizer is caused. At the same time, the application and loss of large amounts of nitrogen fertilizer also leads to a series of serious ecological problems. The nitrogen fertilizer flowing into rivers and lakes causes serious water eutrophication, so that algae are rapidly propagated, the water quality is deteriorated, and further, the fishery is indirectly harmed. In order to deal with water eutrophication and improve water quality, china invests a large amount of manpower and material resources every year to prevent and treat pollution, and local financial burden is brought.
Nitrogen fixation in symbiosis with rhizobia is a unique functional attribute of leguminous plants. The rhizobia has strong nitrogen fixation capacity, and the total amount of biological nitrogen fixation per year on the earth surface is about 10 according to statistics 8 t, wherein the nitrogen fixation amount of rhizobia in leguminous plants is about 5.5X 10 7 t, accounting for about 55% of the total biological nitrogen fixation. Therefore, the research on the plant root nodule forming mechanism is clear, and the method has great economic and social values by improving the number of root nodules or the nitrogen fixation efficiency of the root nodules and applying the method to agricultural production.
Studies in Medicago truncatula, lotus corniculatus and Glycine max show that CLAVATA3/ESR (CLE) -RELATED-ROOT SIGNAL1 (CLE-RS 1), CLE-RS2 and CLE-RS3 in CLAVATA3/ESR (CLE) small peptide are negative regulators of nodule formation, and that their deletion mutants can both lead to different degrees of increase in the number of nodules in leguminous plants. Expression of these small peptides is induced by expression of the NODULE INCEPTION (NIN) and NIN-like protein NITRATE UNRESPONSIVE SYMBIOSIS1 (NRSYM 1) genes. Meanwhile, glycosyltransferases LjPLENTY, mtRDN1 and PsNOD are involved in the glycosylation process of the small peptides. This glycosylation process has an important role in the biological activity of small peptides. In addition, plant hormones such as auxin, cytokinin, gibberellin, ethylene and the like are also widely involved in the nodulation of leguminous plants. Although there have been reports on the function of downstream genes in the process of nodule formation, there are few reports on the influence of upstream transcription factor genes on the number of nodules through the regulation of a series of downstream genes.
Disclosure of Invention
In view of the above prior art, in order to study the change of the number of nodules of leguminous plants by means of genetic engineering techniques, the present invention found two genes that regulate the number of nodules and provided a method for knocking out the genes to obtain transgenic leguminous plants with increased number of nodules.
The invention is realized by the following technical scheme:
application of HAN homologous gene of leguminous plants in regulating and controlling the number of nodules of leguminous plants. The HAN homologous gene of the leguminous plant is selected from MtHAN1 gene and MtHAN2 gene, the nucleotide sequence of the MtHAN1 gene is shown as SEQ ID No.1, and the nucleotide sequence of the MtHAN2 gene is shown as SEQ ID No. 2.
The leguminous plant is selected from alfalfa, medicago truncatula, clover, soybean and peanut.
Application of HAN homologous gene of leguminous plant in preparing transgenic leguminous plant is provided. In the specific application, the HAN homologous genes (MtHAN 1 gene and MtHAN2 gene) in seeds or plants of leguminous plants are knocked out, so that transgenic leguminous plants are obtained, and the root nodule quantity of the transgenic leguminous plants is higher than that of wild leguminous plants.
A method for obtaining transgenic leguminous plants by knocking out the HAN homologous gene of leguminous plants comprises the following steps: knocking out HAN homologous genes (MtHAN 1 gene and MtHAN2 gene) in seeds or plants of the leguminous plants so as to obtain transgenic leguminous plants, wherein the number of root nodules is higher than that of wild leguminous plants.
The invention separates out the knockout mutant strain of the MtHAN1 and MtHAN2 genes of the homologous gene family members of the medicago truncatula hance HAN for the first time, and experiments prove that the gene participates in regulating and controlling the quantity of the root nodules of the leguminous plants, and the transgenic leguminous plants with the root nodules higher than that of wild plants can be obtained by knocking out the gene.
The invention utilizes specific primers (nucleotide sequences are shown as SEQ ID No.5, 6, 7 and 8) to clone MtHAN1 and MtHAN2 genes from medicago truncatula by RT-PCR technology, utilizes the constructed mutant population to screen out knockout mutant strains of the MtHAN1 and MtHAN2 genes, and obtains double mutant plants of MtHAN1MtHAN2 by a hybridization method. Wild type R108 and mthan1mthan2 double mutant seeds are planted in sand after germinating for 5 days at 4 ℃: in the seedling raising holes with perlite as 1. After 3 weeks the nodule morphology was observed and the number of nodules of the wild type and the double mutant respectively was counted. The number of nodules of 37 wild-type and 41 double-mutant plants were counted in the experiment. Statistics show that the root nodule morphology of the wild type plant and the mutant plant has no obvious difference, but the number of the root nodules is obviously different. The number of wild type plant nodules averaged about 13, while the number of double mutant plant nodules averaged about 20. Compared with the wild type, the double mutant plant has the advantage that the root nodule number is remarkably increased. This demonstrates that these two genes are involved in regulating nodulation in leguminous plants. The gene is knocked out, the number of the root nodules of the species can be obviously increased in transgenic leguminous plants, the method indicates that novel leguminous plants which are more beneficial to biological nitrogen fixation can be created after the method is implemented, the method can be used for subsequent leguminous plant variety improvement, and the method has great significance for reducing the use amount of nitrogen fertilizer during leguminous plant planting in China and increasing the crop yield.
The various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.
Drawings
FIG. 1: cloning of medicago truncatula MtHAN1, mtHAN2 genes and identification of mutants, wherein, a: the gene structure of MtHAN1 and MtHAN2 and the insertion position of Tnt1 transposon in the mutant; b: RT-PCR detects the transcription of MtHAN1 and MtHAN2 genes in wild type and mutant. In 2 mutant lines of each of MtHAN1 and MtHAN2, tnt1 is inserted into the exon region of the gene. When the RT-PCR method detects the transcription conditions of MtHAN1 and MtHAN2 in two single mutants, the normal transcript of the gene cannot be detected.
FIG. 2: rhizobium phenotype analysis of the medicago truncatula mthan1mthan2 double mutant, wherein a: root nodule growth when root nodules are induced by wild plants; b: root nodule growth when the double mutant plants induce root nodules.
FIG. 3: counting the change condition of the number of the nodules when wild type plants and double mutant plants induce the nodules, wherein the number of the nodules can be obviously increased when both MtHAN1 and MtHAN2 are mutated.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
EXAMPLE 1 cloning of Medicago truncatula genes MtHAN1, mtHAN2
The sequence of the Arabidopsis HAN gene is obtained through an Arabidopsis database website TAIR, and then sequence alignment search is carried out by utilizing BLAST to obtain the genome sequences (shown as SEQ ID No.1 and 2) of MtHAN1 and MtHAN2, and the amino acid sequences of the expressed protein are shown as SEQ ID No.3 and 4. Primers shown in SEQ ID Nos. 5 and 6 (HAN 1-RT-F, HAN 1-RT-R) and SEQ ID Nos. 7 and 8 (HAN 2-RT-F, HAN 2-RT-R) were designed based on the sequences. The TRIzol kit is used for extracting RNA of wild leaf tissue of the medicago truncatula, and the full-length CDS sequence of MtHAN1 and MtHAN2 genes is amplified by an RT-PCR method. The gene sequence was ligated into pEARLEYGATE103 vector using Gateway technology and then sequenced to verify the correctness of the cloned sequence.
Example 2 obtaining and identifying mutants of Medicago truncatula MtHAN1, mtHAN2
A mutant library marked by Tnt1 of the medicago truncatula is screened by utilizing a Thermal asymmetric interlaced-PCR (TAIL-PCR) technology, and two mutant strains with Tnt1 inserted into MtHAN1 and MtHAN2 genes are respectively screened from 22000 mutant strains (figure 1A). Molecular biological identification results showed that, in all four mutant lines, tnt1 was inserted into the second exon region of the MtHAN1, mtHAN2 genes, resulting in abnormal transcription of the genes (fig. 1B).
Example 3 Rhizobium phenotype analysis of alfalfa MtHAN1, mtHAN2 gene mutant plants
After germination, inoculating sinorhizobium to the wild R108 and mthan1mthan2 double mutant seeds of medicago truncatula (after germination of the wild R108 and mthan1mthan2 double mutant seeds at 4 ℃ for 5 days, the seeds are planted in seedling culture holes with sand: perlite being 1. The result shows that the simultaneous knockout of MtHAN1 and MtHAN2 genes does not affect the root nodule morphogenesis, but can obviously increase the root nodule generation number of plants. The number of nodules of 37 wild-type and 41 double-mutant plants were counted in the experiment. Statistics show that the number of wild type plant nodules is about 13 on average, and the number of double mutant nodules is about 20 on average. There was a significant increase in the number of nodules in the double mutant plants relative to the wild type (fig. 2A, B, fig. 3).
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Sequence listing
<110> name of applicant
<120> application of HAN homologous gene of leguminous plant in regulation and control of root nodule number of leguminous plant
<141> 2019-10-29
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Claims (3)
1. The application of the HAN homologous gene of the leguminous plant to the increase of the number of the root nodules of the leguminous plant is characterized in that: the HAN homologous gene of the leguminous plant is MtHAN1 gene and MtHAN2 gene, the nucleotide sequence of the MtHAN1 gene is shown as SEQ ID No.1, and the nucleotide sequence of the MtHAN2 gene is shown as SEQ ID No. 2; the leguminous plant is selected from medicago truncatula.
2. The application of the knockout leguminous plant HAN homologous gene in the preparation of transgenic leguminous plants is characterized in that: the HAN homologous gene of the leguminous plant is MtHAN1 gene and MtHAN2 gene, the nucleotide sequence of the MtHAN1 gene is shown as SEQ ID No.1, and the nucleotide sequence of the MtHAN2 gene is shown as SEQ ID No. 2; in the specific application, the MtHAN1 gene and the MtHAN2 gene in seeds or plants of the leguminous plants are knocked out, so that the transgenic leguminous plants are obtained, and the number of root nodules of the transgenic leguminous plants is higher than that of wild leguminous plants; the leguminous plant is selected from medicago truncatula.
3. A method for obtaining transgenic leguminous plants by knocking out the HAN homologous gene of leguminous plants, characterized in that: knocking out HAN homologous genes in seeds or plants of the leguminous plants so as to obtain transgenic leguminous plants, wherein the root nodule quantity of the transgenic leguminous plants is higher than that of wild leguminous plants; the HAN homologous gene of the leguminous plant is MtHAN1 gene and MtHAN2 gene, the nucleotide sequence of the MtHAN1 gene is shown as SEQ ID No.1, and the nucleotide sequence of the MtHAN2 gene is shown as SEQ ID No. 2; the leguminous plant is selected from medicago truncatula.
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| CN113429467B (en) * | 2020-03-20 | 2023-01-24 | 中国科学院微生物研究所 | Application of NPF7.6 protein in regulation and control of nitrogen tolerance of leguminous plant root nodule |
| CN114478728B (en) * | 2020-11-13 | 2023-07-21 | 中国科学院微生物研究所 | Application of nKCBP protein in regulation and control of nitrogen fixation capacity of leguminous plants |
| CN114085852B (en) * | 2021-06-10 | 2024-02-13 | 中国科学院西双版纳热带植物园 | CLL1 gene for regulating plant semi-dwarf plant type and leaf ratio and application of orthologous gene of leguminous plant |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000022138A1 (en) * | 1998-10-14 | 2000-04-20 | Agriculture And Agri-Food Canada | Genetic factor of s. meliloti usda 1170 containing nodulation efficiency factor |
| CN108586592A (en) * | 2018-04-23 | 2018-09-28 | 中国科学院上海生命科学研究院 | The gene for regulating and controlling the nodule number of root nodule plant and its application in terms of high-efficiency nitrogen-fixing |
| CN108624596A (en) * | 2018-05-04 | 2018-10-09 | 华南农业大学 | It is a kind of regulation and control Legume nodule growth gene GmSPX5 and its application |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000022138A1 (en) * | 1998-10-14 | 2000-04-20 | Agriculture And Agri-Food Canada | Genetic factor of s. meliloti usda 1170 containing nodulation efficiency factor |
| CN108586592A (en) * | 2018-04-23 | 2018-09-28 | 中国科学院上海生命科学研究院 | The gene for regulating and controlling the nodule number of root nodule plant and its application in terms of high-efficiency nitrogen-fixing |
| CN108624596A (en) * | 2018-05-04 | 2018-10-09 | 华南农业大学 | It is a kind of regulation and control Legume nodule growth gene GmSPX5 and its application |
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